Screening Of The LRP16 Interacting Proteins And The Subsequent Function Regulation

Objectives:①To screen LRP 16 interaction proteins from cDNA library of MCF-7 human breast cancer cell line by yeast two-hybrid technology;②To confirm the interaction of LRP16 with nuclear receptors and NF-kB;③To study the regulation of LRP16 on androgen receptor(AR)transactivation and the effects of LRP16 on the proliferation of prostate cancer cell line LNCap in different androgen levels;④To investigate the regulation of androgen on the expression of LRP16.Methods:①The LRP16 fragment that can encode full-length protein sequence was inserted into bait vector pGBKT7 of yeast two-hybrid system at the EcoR I sites.Following the recombined construct was transformed into yeast cells AH109 and Y187,respectively.The toxicity and the self-activating transcriptional activation of human LRP16 protein in these two yeast cells were firstly detected.The ectopic expression of human LRP16 protein in the total yeast protein extracts was also measured by western blot analysis.②A yeast two-hybrid screening was performed by cotransformation of the ds cDNA and linearized pGADT7-Rec with the bait pGBKT7-LRP16 to the yeast AH109 plating on quadruple dropout medium.The Inserted fragments of identified positive clones were amplified and sequenced.And simple libarary plasmids of positive clones were obtained from competent bacterium coli Top10 transformated with yeast plasmid.Following,the interactions of LRP16 and positive plasmid were tested in yeast cells again.③The interaction between LRP16 and ART-27 was confirmed by GST pull-down in vitro assay and co-immunoprecipitation analysis in vivo.And the direct interactions of LRP16 with some nuclear receptors and NF-kB were further confirmed by GST pull-down.In addition,we checked the interacting domains of both LRP16 and AR.④Luciferase report gene assays were used to test the effects of LRP16 on AR transactivation and regulation of AR on LRP16 promotors.⑤MTT was used to investigate effects of down-regulation of endogenous LRP16 on proliferation of LNCap induced by different androgen levels⑥The expression of LRP16 was determined by Western blot in the presence of different testosterone levels and different androgen-induced time.Also,we investigated LRP16 expression in different prostate cancer cell line.Results:①The insertion direction and size of human LRP16 in the recombined plasmid were both correct,the toxicity and self-activating transcriptional activation of human LRP16 protein were not observed in yeast cells.Importantly,the human LRP16 protein can be effectively expressed in transformed yeast.②There were 8 different candidate gene sequences obtaind by Blast analysis in NCBI.Four kinds of putative positive library plasmids were further tested interaction in yeast by transformation and 3/4 could group yeast clone.③LRP16 has direct interactions with not only ART-27 but also NF-kB and nuclear receptors including AR,ERβ,PPARα,PPARγ.④The unique domain in C terminal of LRP 16 interacts with LBD of AR;⑤In the presence of testosterone,overexpression of LRP16 enhances AR transcriptional activity. Conversely,downregulation of endogenous LRP16 expression by RNA interference suppresses AR transcriptional activity.⑥Depletion of LRP16 protein level results in inhibition of LNCap froliferation in difference androgen levels.⑦Expression of LRP16 are increased by low androgen level and testosterone inducing for 3 hours.And AR can enhance promotors activity of LRP16.⑧AR positive cell line LNCap has more LRP16 protein than AR negative cell line DU145.Conclusions:①We obtained a novel class of LRP16 interacting proteins by yeast two-hybrid system.②LRP16 has direct interactions with AR and NF-kB independing on ART-27.③LRP16 is interacting protein of some nuclear receptors.④LRP16 which was a target gene of androgen can feedbackly enhance the transcriptional activition of AR,and it is a co-activator of AR.⑤LRP16 regulates proliferation of androgen responsive prostate cancer cell possibly by interaction of LRP16 and AR.